During the second year of the project there has not been a major shift in the specific aims, but we have modified one and added another. The major goal is still developing a better understanding of the gas phase structures of metalated peptides and using this information as a foundation for characterizing, predicting, and exploiting the fragmentation behavior of these species in the analysis of peptide sequences. The specific aims from the original proposal are restated here (including the modification to number 4) along with the new area (number 6). 1. Because the investigation of the fragmentation behavior of metalated peptides has been hampered by a lack of structural data, we will develop a force-field approach for predicting the most likely coordination sites on peptides. This computational model will play a role in guiding and interpreting subsequent experiments. 2. The strengths of metal peptide interactions will be evaluated for a series of alkali mental/dipeptide complexes. For these studies, Cooks' kinetic method will be used to develop a relative cation affinity scale for the dipeptides and the results will be compared to predictions from the computational model. 3. We will use our structural information about metalated peptides to investigate the role that lithium plays in catalyzing the C-terminal fragmentation of peptides. This will also involve further studies of the scope and limitations of this analytically useful fragmentation pathway. 4. The fragmentation behavior of alkaline earth metal/peptide complexes, [peptide -H+ + M++]+ will be evaluated in an effort to further develop the analytical value of these species. To support these studies, our force- field approach will be extended to include complexes of this type. 5. The effect of post-translational modifications on the fragmentations of metal/peptide complexes will be evaluated for N-terminal acylation and serine phosphorylation. Although it is possible that we will not be able to complete this portion of the project during the grant period, a foundation will be developed for subsequent studies. 6. We will investigate multiply charged, anionic peptides as tools for peptide sequencing. In particular, peptides with aspartate and glutamate residues will be treated with various electrophiles in an effort to find systems that either identify the location of the acidic residue or cause selective cleavage of a peptide bond near the acidic residue.